Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment.

Background & Aims: Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3). Methods: PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining. Results: PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates. Conclusions: Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness. Impact and implications: Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

cccDNA Maintenance in Chronic Hepatitis B - Targeting the Matrix of Viral Replication.

Chronic hepatitis B is a numerically important cause of cirrhosis and hepatocellular carcinoma, despite an effective prophylactic vaccine and well-tolerated and effective oral antivirals. Both the incapacity of the immune system to clear hepatitis B virus (HBV) infection and the unique replication strategies adopted by HBV are considered key determinants of HBV chronicity. In this regard, the formation of the HBV DNA minichromosome, the covalently closed circular DNA (cccDNA), in the nucleus of infected hepatocytes, is essential not only for the production of all viral proteins but also for HBV persistence even after long-term antiviral therapy. Licensed polymerase inhibitors target the HBV reverse transcriptase activity, control the disease with long-term therapy but fail to eliminate the cccDNA. Consequently, the production of viral RNAs and proteins, including the hepatitis B surface antigen (HBsAg), is not abolished. Novel therapeutic efforts that are in the pipeline for early clinical trials explore novel targets and molecules. Such therapeutic efforts focus on achieving a functional cure, which is defined by the loss of HBsAg and undetectable HBV DNA levels in serum. Since a true cure of HBV infection requires the elimination of the cccDNA from infected cells, comprehension of the mechanisms implicated in cccDNA biogenesis, regulation and stability appears necessary to achieve HBV eradication. In this review, we will summarize the state of knowledge on cccDNA metabolism, focusing on insights suggesting potential weak points of the cccDNA that may be key for the development of therapeutic approaches and design of clinical trials aiming at lowering cccDNA loads and activity.

Treatment with tenofovir disoproxil fumarate or entecavir in chronic hepatitis B virus-infected patients with renal impairment: results from a 7-year, multicentre retrospective cohort study.

BACKGROUND: Limited data exist regarding tenofovir disoproxil fumarate (TDF) safety and effectiveness in chronic hepatitis B virus-infected (CHB) patients with renal impairment (RI). AIMS: To compare real-world data on renal safety and effectiveness of TDF vs entecavir (ETV) in CHB patients with moderate-to-severe RI. METHODS: Retrospective, non-interventional, cohort study analysing medical records for TDF/ETV-treated CHB patients (54 European centres). Included patients experienced moderate-to-severe RI (creatinine clearance 20-60 mL/min [Cockcroft-Gault]) either before TDF/ETV initiation ('before' subgroup [baseline = treatment initiation]) or after TDF/ETV initiation ('after' subgroup [baseline = first RI occurrence]). The primary objective was TDF safety, particularly renal-related adverse events of special interest (AESI). TDF and ETV safety and effectiveness were compared and multivariate analyses were performed using inverse probability treatment weighting. RESULTS: 'Before' subgroup included 107 TDF- and 91 ETV-treated patients; 'after' subgroup included 212 TDF- and 77 ETV-treated patients. Mean baseline creatinine clearance was higher for TDF- vs ETV-treated patients (both subgroups). Median follow-up was 3.1 years (both treatments). AESI were more frequent with TDF vs ETV ('before': 18.7% vs 8.8%; 'after': 9.9% vs 3.9%); however, differences were not significant by multivariate analysis. Only TDF-treated patients experienced renal tubular dysfunction (6.5% 'before'; 1.9% 'after') as well as renal adverse events leading to treatment discontinuation (8.4% 'before'; 7.1% 'after'). Effectiveness was similar between treatments. CONCLUSIONS: Overall safety was similar for TDF vs ETV (both subgroups). Given that renal tubular dysfunction occurred with TDF and not with ETV, renal safety concerns may be greater with TDF in CHB patients with RI.

Hepatitis delta virus persists during liver regeneration and is amplified through cell division both in vitro and in vivo.

OBJECTIVE: Hepatitis delta virus (HDV) was shown to persist for weeks in the absence of HBV and for months after liver transplantation, demonstrating the ability of HDV to persevere in quiescent hepatocytes. The aim of the study was to evaluate the impact of cell proliferation on HDV persistence in vitro and in vivo. DESIGN: Genetically labelled human sodium taurocholate cotransporting polypeptide (hNTCP)-transduced human hepatoma(HepG2) cells were infected with HBV/HDV and passaged every 7 days for 100 days in the presence of the entry inhibitor Myrcludex-B. In vivo, cell proliferation was triggered by transplanting primary human hepatocytes (PHHs) isolated from HBV/HDV-infected humanised mice into naïve recipients. Virological parameters were measured by quantitative real time polymerase chain reaction (qRT-PCR). Hepatitis delta antigen (HDAg), hepatitis B core antigen (HBcAg) and cell proliferation were determined by immunofluorescence. RESULTS: Despite 15 in vitro cell passages and block of viral spreading by Myrcludex-B, clonal cell expansion permitted amplification of HDV infection. In vivo, expansion of PHHs isolated from HBV/HDV-infected humanised mice was confirmed 3 days, 2, 4 and 8 weeks after transplantation. While HBV markers rapidly dropped in proliferating PHHs, HDAg-positive hepatocytes were observed among dividing cells at all time points. Notably, HDAg-positive cells appeared in clusters, indicating that HDV was transmitted to daughter cells during liver regeneration even in the absence of de novo infection. CONCLUSION: This study demonstrates that HDV persists during liver regeneration by transmitting HDV RNA to dividing cells even in the absence of HBV coinfection. The strong persistence capacities of HDV may also explain why HDV clearance is difficult to achieve in HBV/HDV chronically infected patients.

Mechanism of Hepatitis B Virus Persistence in Hepatocytes and Its Carcinogenic Potential.

Liver disease associated with persistent infection with hepatitis B virus (HBV) continues to be a major health problem of global impact. Despite the existence of an effective vaccine, at least 240 million people are chronically infected worldwide, and are at risk of developing liver cirrhosis and hepatocellular carcinoma. Although chronic HBV infection is considered the main risk factor for liver cancer development, the molecular mechanisms determining persistence of infection and long-term pathogenesis are not fully elucidated but appear to be multifactorial. Current therapeutic regimens based on the use of polymerase inhibitors can efficiently suppress viral replication but are unable to eradicate the infection. This is due both to the persistence of the HBV genome, which forms a stable minichromosome, the covalently closed circular DNA (cccDNA), in the nucleus of infected hepatocytes, as well as to the inability of the immune system to efficiently counteract chronic HBV infection. In this regard, the unique replication strategies adopted by HBV and viral protein production also appear to contribute to infection persistence by limiting the effectiveness of innate responses. The availability of improved experimental systems and molecular techniques have started to provide new information about the complex network of interactions that HBV establishes within the hepatocyte and that may contribute to disease progression and tumor development. Thus, this review will mostly focus on events involving the hepatocyte: the only target cell where HBV infection and replication take place.

Binding of hepatitis B virus to its cellular receptor alters the expression profile of genes of bile acid metabolism.

UNLABELLED: Chronic hepatitis B virus (HBV) infection has been associated with alterations in lipid metabolism. Moreover, the Na+-taurocholate cotransporting polypeptide (NTCP), responsible for bile acid (BA) uptake into hepatocytes, was identified as the functional cellular receptor mediating HBV entry. The aim of the study was to determine whether HBV alters the liver metabolic profile by employing HBV-infected and uninfected human liver chimeric mice. Humanized urokinase plasminogen activator/severe combined immunodeficiency mice were used to establish chronic HBV infection. Gene expression profiles were determined by real-time polymerase chain reaction using primers specifically recognizing transcripts of either human or murine origin. Liver biopsy samples obtained from HBV-chronic individuals were used to validate changes determined in mice. Besides modest changes in lipid metabolism, HBV-infected mice displayed a significant enhancement of human cholesterol 7α-hydroxylase (human [h]CYP7A1; median 12-fold induction; P<0.0001), the rate-limiting enzyme promoting the conversion of cholesterol to BAs, and of genes involved in transcriptional regulation, biosynthesis, and uptake of cholesterol (human sterol-regulatory element-binding protein 2, human 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and human low-density lipoprotein receptor), compared to uninfected controls. Significant hCYP7A1 induction and reduction of human small heterodimer partner, the corepressor of hCYP7A1 transcription, was also confirmed in liver biopsies from HBV-infected patients. Notably, administration of Myrcludex-B, an entry inhibitor derived from the pre-S1 domain of the HBV envelope, provoked a comparable murine CYP7A1 induction in uninfected mice, thus designating the pre-S1 domain as the viral component triggering such metabolic alterations. CONCLUSION: Binding of HBV to NTCP limits its function, thus promoting compensatory BA synthesis and cholesterol provision. The intimate link determined between HBV and liver metabolism underlines the importance to exploit further metabolic pathways, as well as possible NTCP-related viral-drug interactions.

Virological response to entecavir is associated with a better clinical outcome in chronic hepatitis B patients with cirrhosis.

OBJECTIVE: Entecavir (ETV) is a potent inhibitor of viral replication in chronic hepatitis B and prolonged treatment may result in regression of fibrosis. The aim of this study was to investigate the effect of ETV on disease progression. DESIGN: In a multicentre cohort study, 372 ETV-treated patients were investigated. Clinical events were defined as development of hepatocellular carcinoma (HCC), hepatic decompensation or death. Virological response (VR) was defined as HBV DNA <80 IU/ml. RESULTS: Patients were classified as having chronic hepatitis B without cirrhosis (n=274), compensated cirrhosis (n=89) and decompensated cirrhosis (n=9). The probability of VR was not influenced by severity of liver disease (p=0.62). During a median follow-up of 20 months (IQR 11-32), the probability of developing clinical events was higher for patients with cirrhosis (HR 15.41 (95% CI 3.42 to 69.54), p<0.001). VR was associated with a lower probability of disease progression (HR 0.29 (95% CI 0.08 to 1.00), p=0.05) which remained after correction for established risk factors such as age. The benefit of VR was only significant in patients with cirrhosis (HR 0.22 (95% CI 0.05 to 0.99), p=0.04) and remained after excluding decompensated patients (HR 0.15 (95% CI 0.03 to 0.81), p=0.03). A higher HBV DNA threshold of 2000 IU/ml was not associated with the probability of disease progression (HR 0.20 (95% CI 0.03 to 1.10), p=0.10). CONCLUSION: VR to ETV is associated with a lower probability of disease progression in patients with cirrhosis, even after correction for possible baseline confounders. When using a threshold of 2000 IU/ml, the association between viral replication and disease progression was reduced, suggesting that complete viral suppression is essential for nucleoside/nucleotide analogue treatment, especially in patients with cirrhosis.

Virion half-life in chronic hepatitis B infection is strongly correlated with levels of viremia.

UNLABELLED: Analysis of hepatitis B virus (HBV) kinetics with mathematical models may disclose new aspects of HBV infection and host response mechanisms. To determine the kinetics of virion decay from the blood of patients in different phases of chronic infection, we applied mathematical modeling to real-time polymerase chain reaction assays, which enable quantification of viremia and intrahepatic HBV productivity by measuring both copy number and activity of covalently closed circular DNA (relaxed circular DNA/covalently closed circular DNA) in the liver of 80 untreated chronically active HBV carriers (38 hepatitis B e antigen [HBeAg]-positive and 42 HBeAg-negative individuals). We found that the half-life of circulating virions is very fast (median 46 and 2.5 minutes in HBeAg-positive and HBeAg-negative individuals, respectively) and strongly related to viremia, with clearance rates significantly accelerating as viral loads decrease. To investigate whether immune components can influence the kinetics of virion decay, we analyzed viral dynamics in immunodeficient urokinase-type plasminogen activator chimera mice. Virion half-life in mice (range, 44 minutes to >4 hours) was comparable to estimates determined in high viremic carriers, implying that clearance rates in these patients are mostly determined by common nonspecific mechanisms. Notably, the lack of correlation between virion half-life and viremia in mice indicated that immune components significantly accelerate virion clearance rates in individuals with low titers. CONCLUSION: Our analyses suggest that both host defense mechanisms and levels of circulating virions affect the kinetics of HBV decay assessed in the serum of chronic carriers. Identification of the factors affecting clearance rates will be important for future antiviral drug developments and it may give insights into the mechanisms involved in clearance of other chronic infections, such as human immunodeficiency virus and hepatitis C virus.

Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein.

360 million people are chronically infected with the human hepatitis B virus (HBV) and are consequently prone to develop liver cirrhosis and hepatocellular carcinoma. As approved therapeutic regimens-which modulate patients' antiviral defenses or inhibit the viral reverse transcriptase-are generally noncurative, strategies interfering with other HBV replication steps are required. Expanding on our demonstration that acylated peptides derived from the large HBV envelope protein block virus entry in vitro, we show their applicability to prevent HBV or woolly monkey hepatitis B virus infection in vivo, using immunodeficient urokinase-type plasminogen activator (uPA) mice repopulated with primary human or Tupaia belangeri hepatocytes. Accumulation of the peptides in the liver, their extraordinary inhibitory potency and specific mode of action permit subcutaneous delivery at very low doses. Inhibition of hepadnavirus entry thus constitutes a therapeutic approach to prevent primary HBV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients.

Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients.

BACKGROUND & AIMS: Knowledge of factors regulating transcriptional activity of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may help in understanding mechanisms of viral decay and how these processes are thwarted in chronically HBV-infected patients. METHODS: Liver biopsies from 119 treatment-naive chronically infected patients (42 HBeAg-positive and 77 HBeAg-negative) were determined for HBV transcriptional and replicative activity. RESULTS: Significantly lower median serum HBV DNA (-4 log), intrahepatic HBV DNA (-2 log), and cccDNA (-1 log) amounts were measured in HBeAg-negative versus HBeAg-positive patients. Despite a good correlation found between intrahepatic amounts of progeny virions and serum HBV DNA in all patients, cccDNA levels did not correlate with serum titers in HBeAg-negative individuals. Analysis of HBV RNA transcripts showed that impaired virion productivity in HBeAg-negative individuals was due to lower steady-state levels of pregenomic RNA produced per cccDNA. Interestingly, preS/S RNA levels and serum HBsAg concentrations did not differ between HBeAg-positive and HBeAg-negative patients when normalized for cccDNA contents, showing that subviral particle production was not impaired in HBeAg-negative patients and correlated with cccDNA levels. Although the majority of HBeAg-negative individuals harbored cccDNA with common precore and/or basal core promoter mutations, occurrence of these variants was not responsible for reduced viral replication. Instead, replacement of wild-type cccDNA with core promoter mutants reestablished high virion productivity. CONCLUSIONS: Lower viremia in HBeAg-negative individuals is not only due to lower cccDNA content but also to impaired virion productivity, which can arise without emergence of HBeAg variants and without affecting HBsAg production.

Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is responsible for persistent infection of hepatocytes. The aim of this study was to determine changes in intrahepatic cccDNA in patients with chronic hepatitis B (CH-B) during 48 weeks of antiviral therapy and its correlation to virological, biochemical, and histological parameters. Twenty-six HBsAg-positive CH-B patients received combination treatment with pegylated interferon alpha-2b (peg-IFN) and adefovir dipivoxil (ADV) for 48 weeks. Paired liver biopsies from before and at the end of treatment were analyzed for intrahepatic HBV-DNA. Median serum HBV-DNA had decreased by -4.9 log10 copies/mL at the end of treatment and was undetectable in 13 individuals (54%). Median intrahepatic total HBV-DNA and cccDNA had decreased by -2.2 and -2.4 log10, respectively. Changes in intracellular HBV-DNA positively correlated with HBsAg serum reduction and were accompanied by a high number of serological responders. Eight of 15 HBeAg-positive patients lost HBeAg, and five developed anti-HBe antibodies during treatment. These eight patients exhibited lower cccDNA levels before and at the end of therapy than did patients without HBeAg loss. Four patients developed anti-HBs antibodies. ALT normalized in 11 patients. The number of HBs-antigen- and HBc-antigen-positive hepatocytes was significantly lower after treatment, suggesting the involvement of cytolytic mechanisms. In conclusion, combination therapy with peg-IFN and ADV led to marked decreases in serum HBV-DNA and intrahepatic cccDNA, which was significantly correlated with reduced HBsAg.

Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly(ADP-ribosyl)ation.

Chronic infection with hepatitis B virus (HBV) is associated with an increased risk for the development of cirrhosis and hepatocellular carcinoma (HCC). Although clonal HBV DNA integrations are detected in nearly all HCCs the role of these integrations in hepatocarcinogenesis is poorly understood. We have used a cloning protocol that allows studying the frequency and the natural history of HBV DNA integrations in cell culture. Southern blot analysis of the genomic DNA of HepG2 2.2.15 subclones, which replicate HBV, enabled us to detect new HBV DNA integrations in approximately 10% of the HepG 2.2.15 subclones over 4 rounds of sequential subcloning, whereas no loss of any preexisting HBV DNA integrations was observed. Treatments of HepG2 cells with H(2)O(2), designed to increase DNA damage, increased the frequency of HBV integrations to approximately 50% of the subclones and treatments designed to inhibit DNA repair, by inhibiting Poly(ADP-ribosyl)ation, also increased the frequency of HBV integration to 50%. These findings suggest that DNA strand breaks induced by oxidative stress during persistent HBV infection in humans may increase HBV DNA integration events, whereas PARP-1 activity may function to limit the occurrence of de novo HBV DNA integrations.